Abstract 2161: In vitro osteoblast assays for studying the effects of cancer therapeutics on bone biology

Abstract

Abstract Bone cell activity is an important factor in regulating bone metastases from tumor cells in the skeletal environment. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Bone then releases stimulatory factors that in turn promote the growth and proliferation of cancer cells. This process is called the vicious cycle often leading to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. Osteolytic lesions are common in breast and lung cancer and multiple myeloma. There is a vicious cycle between cancer cells and bone cells also in the context of osteoblastic lesions. In osteoblastic metastases, the vicious cycle observed in osteolytic disease takes place but in addition to this cancer cells produce osteoblast-stimulating factors including bone morphogenetic protein (BMP), epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Osteoblasts also influence osteoclasts by producing RANKL, which stimulates osteoclast differentiation. Many cancer patients with bone metastasis have both osteolytic and osteoblastic lesions. This is common for example in prostate cancer. Our aim was to establish an in vitro cell culture model to study the effects of compounds on osteoblast differentiation and activity. A mouse osteoblast progenitor cell line KS483 was used in the study. BMP-2 was used as a test compound and was added in the beginning of culture and simultaneously with culture medium change every 3-4 days. Ascorbic acid was added into the cell culture medium at day 4. In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8 from cell lysates. Total protein content was measured from the same samples. In the osteoblast activity assay, KS483 mouse osteoprogenitor cells were cultured for 13 days, during which N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to demonstrate effects on organic bone matrix formation. β-glycerophosphate was added to the culture at day 11. The cultures were stopped at day 13 by removing the culture media from the wells and adding hydrochloric acid. Calcium deposition, a marker of inorganic bone formation, was determined at the end of the study. BMP-2 stimulated osteoblast differentiation and activity shown by the increase in ALP, PINP and calcium levels. The results suggest that the KS483 cell line can be used for setting up reliable in vitro models of osteoblast differentiation and activity. These osteoblast assays can be used for studying the effects of compounds on bone formation and cancer related bone events. Citation Format: Jenni H. Mäki-Jouppila, Jussi M. Halleen, Katja Fagerlund. In vitro osteoblast assays for studying the effects of cancer therapeutics on bone biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2161.